Methylenetetrahydrofolate reductase (MTHFR) is a protein that is important in folate metabolism. Its enzymatic activity is to convert 5, 10-methylenetetrahydrofolate to 5'-methyl tetrahydrofolate, which in turn is important in the methylation of homocysteine to methionine. Several variants and mutations in the gene for MTHFR have been described. Moderate or severe deficiencies of this enzyme lead to a number of disorders including hyperhomocystenuria and homocystenuria. The clinical outcome of the deficiency include peripheral neuropathy, developmental delay, hypotonia and seizures [1]. Mild forms of the reduced MTHFR activity are present in high frequencies in the general population. These variant forms of these enzymes lead to mild homocystinuria. High level of homocysteine is a risk factor for arterial disease [2-4]. The genes for human and mouse MTHFR have been isolated and characterized [4-7]. The human gene encodes a protein 656 amino acids in length and the mouse gene encodes a 654 aa protein. There is a high level of conservation between the human and mouse genes. At the amino acid level, the two proteins are 90% identical [5]. The exon-intron organization of the two genes is also identical. Both genes have 11 exons. Although several different size transcripts have been described [6, 7], all of them encode the identical protein. Several variants of the human MTHFR gene have been described. Of these, the C677T variant that converts an alanine codon to valine is the most studied. This is a very common polymorphism and has an allele frequency of approximately 35% in the North American population. The amino acid change leads to a thermolabile enzyme that causes a predisposition of homocystenuria when folate levels are low [4, 8, 9]. In this developmental project, we propose to make mice that have a null allele of the Mthfr gene and a second mouse that carries the homologous C677T variation. Based on the high level of identity between the two proteins and the complete conservation of the 10 amino acids on either side of the variation in the mouse and human genes suggests that the mouse will truly mimic the functional aspects of the C677T polymorphism in humans. Availability of these mice would help us in better defining the role of MTHFR and C677T variant on folate metabolism and how these genetic changes would affect colon cancer susceptibility under different nutritional folate levels. The steps that are involved in generating the mice are as follows: 1. Isolation of a mouse BAC from the 129/SvEvTac strain genomic library, 2. Modification of the gene, 3. Transfection of mouse ES cells with the gene modification construct and screening the colonies for the desired modification, 4. Injection of the modified ES cells into blastocysts to generate chimeras and 5. Mate the chimeras for germ-line transmission of the modified allele.